Bacillus thuringiensis isolates selectively active against certain coleopteran pests

ABSTRACT

The subject invention concerns Bacillus thuringiensis microbes with activity against select coleopteran pests e.g., Diabrotica sp., Hypera sp., and various flea beetles. For example, the B.t. isolates of the invention are active against alfalfa weevils--AW (Hypera brunneipennis), rape flea beetles--RFB (Phyllotreta cruciferae) and corn rootworms--CRW (Diabrotica undecimpunctata undecimpunctata). Thus, these microbes can be used to control these pests. Further, genes encoding toxins active against these pests can be isolated from the B.t. isolates and used to transform other microbes. The transformed microbes then can be used to control susceptible coleopteran pests.

This is a division of application Ser. No. 07/977,386, filed Nov. 17,1992 now abandoned, which is a division of application Ser. No.07/771,964 filed Oct. 4, 1991, now abandoned.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (B.t.) produces an insect toxin designated asδ-endotoxin. It is synthesized by the B.t. sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insectlarvae, is transformed into biologically active moieties by the insectgut juice proteases. The primary target is insect cells of the gutepithelium, which are rapidly destroyed.

The reported activity spectrum of B.t. covers insect species within theorder Lepidoptera, many of which are major pests in agriculture andforestry. The activity spectrum also includes the insect order Diptera,which includes mosquitos and black flies. See Couch, T. L. (1980)"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978)"Use of Entomogenous Bacteria in Agroecosystems," Developments inIndustrial Microbiology 20:97-104. Krieg, et al., Z. ang. Ent. (1983)96:500-508, describe a B.t. isolate named Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.

In European Patent Application 0 202 739 there is disclosed a novel B.t.isolate active against Coleoptera. It is known as B. thuringiensis var.san diego (B.t.sd.). U.S. Pat. No. 4,966,765 discloses thecoleopteran-active Bacillus thuringiensis isolate B.t. PS86B1. EuropeanPatent Application 0 337 604 also discloses a novel B.t. isolate activeagainst Coleoptera. This isolate is B.t. PS43F.

Coleopteran-active strains, such as B.t.sd., B.t, PS86B1, and B.t.PS43F, can be used to control foliar-feeding beetles. The Coloradopotato beetle (Leptinotarsa decemlineata), for example, is susceptibleto the delta-endotoxin of B.t.sd. and larvae are killed upon ingesting asufficient dose of spore/crystal preparation on treated foliage.

There are a number of other beetles that cause economic damage. Forexample, Chrysomelid beetles such as Flea Beetles and Corn Rootworms andCurculionids such as Alfalfa Weevils are particularly important pests.Flea Beetles include a large number of small leaf feeding beetles thatfeed on the leaves of a number of grasses, cereals and herbs. FleaBeetles include a large number of genera (e.g., Attica, Apphthona,Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Systena,and Phyllotreta). The Flea Beetle, Phyllotreta cruciferae, also known asthe Rape Flea Beetle, is a particularly important pest. Corn rootwormsinclude species found in the genus Diabrotica (e.g., D. undecimpunctataundecimpunctata, D. undecimpunctata howardii, D. longicornis, D.virgifera and D. balteata). Corn rootworms cause extensive damage tocorn and curcubits. The Western Spotted Cucumber Beetle, D.undecimpunctata undecimpunctata, is a pest of curcubits in the westernU.S. Alfalfa weevils (also known as clover weevils) belong to the genus,Hypera (H. postica, H. brunneipennis, H. nigrirostris, H. punctata andH. meles), and are considered an important pest of legumes. The Egyptianalfalfa weevil, H. brunneipennis, is an important pest of alfalfa in thewestern U.S.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns Bacillus thuringiensis (B.t.) isolateswhich are active against Diabrotica sp., Hypera sp., and various generaof flea beetles, as listed above. For example, the B.t. isolates of theinvention are active against alfalfa weevils--AW (Hypera brunneipennis),rape flea beetles--RFB (Phyllotreta cruciferae) and corn rootworms--CRW(Diabrotica undecimpunctata undecimpunctata). These activities weredetermined by tests as disclosed in Example 2.

The subject invention also includes mutants of the above B.t. isolateswhich have substantially the same pesticidal properties as the parentB.t. isolates. Procedures for making mutants are well known in themicrobiological art. Ultraviolet light and nitrosoguanidine are usedextensively toward this end.

Further, the invention also includes the treatment of substantiallyintact B.t. cells, and recombinant cells containing the gene of theinvention, to prolong the pesticidal activity when the substantiallyintact cells are applied to the environment of a target pest. Suchtreatment can be by chemical or physical means, or a combination ofchemical or physical means, so long as the technique does notdeleteriously affect the properties of the pesticide, nor diminish thecellular capability in protecting the pesticide. The treated cell actsas a protective coating for the pesticidal toxin. The toxin becomesavailable to act as such upon ingestion by a target insect.

Still further, the invention includes the genes isolatable from the B.t.isolates which genes encode toxins (proteins) active against theabove-noted pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B--Photographs of standard SDS polyacrylamide gels of B.t.isolates of the invention.

DETAILED DISCLOSURE OF THE INVENTION

The Bacillus thuringiensis isolates of the subject invention have thefollowing common characteristics in their biologically pure form:

Common Characteristics of B.t. Isolates of Invention

Colony morphology--Large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

A comparison of the characteristics of the B. thuringiensis isolates ofthe invention to the characteristics of the known B.t. strains B.thuringiensis var. san diego (B.t.sd.) and B. thuringiensis var.kurstaki (HD-1) is shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Comparison of B.t. isolates against B.t.sd. and B.t. HD-1                     __________________________________________________________________________             B.t.sd B.t.HD-1                                                                             B.t. PS92B                                                                           B.t. PS73J3                                                                          B.t. PS75J1                              __________________________________________________________________________    Type of Inclusion                                                                      square bipyramid                                                                            attached                                                                             amorphic                                                                             amorphic                                          wafer         amorphic                                               Size of Alkali-                                                                        72, 64 68, 130                                                                              80 kDa 64, 75, 79,                                                                          63, 75, 79,                              soluble proteins                                                                       kDa    kDa           81 kDa 81 kDa                                   by SDS-PAGE                                                                   __________________________________________________________________________             B.t. PS86A1                                                                          B.t. PS86Q3                                                                          B.t. PS28Q2                                                                          B.t. PS45B1                                                                          B.t. PS140E2                             __________________________________________________________________________    Type of inclusion                                                                      multiple                                                                             1 long and                                                                           1 long and                                                                           multiple                                                                             multiple                                          attached                                                                             small  lemon                                                                  amorphic                                                      Size of Alkali-                                                                        45, 58 kDa                                                                           58, 62, 98,                                                                          63, 70 kDa                                                                           35, 135, 150                                                                         37, 70,78                                soluble proteins                                                                              135, 155      kDa    kDa                                      by SDA-PAGE     kDa                                                           __________________________________________________________________________             B.t. PS143J                                                                          B.t. PS202U2                                                                         B.t. PS74G1                                                                          B.t. PS98A3                                                                          B.t. PS101Z2                             __________________________________________________________________________    Type of inclusion                                                                      amorphic                                                                             multiple long                                                                        amorphic                                                                             long   long                                     Size of Alkali-                                                                        29, 40, 78, 96,                                                                      45, 58 kD                                                                            61, 115, 150,                                                                        33, 145, 155                                                                         145, 160 kDa                             soluble proteins                                                                       98 kDa        155 kDa                                                                              kDa                                             by SDA-PAGE                                                                   __________________________________________________________________________     NOTE:                                                                         Isolate B.t. PS73J3 is indistinguishable morphologically from B.t. PS75J1     Further tests will finalize identity.                                    

The cultures disclosed in this application have been deposited in theAgricultural Research Service Patent Culture Collection (NRRL), NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604, USA.

    ______________________________________                                        Culture         Repository No.                                                                            Deposit date                                      ______________________________________                                        Bacillus thuringiensis PS92B                                                                  NRRL B-18889                                                                              Sept. 23, 1991                                    Bacillus thuringiensis PS28Q2                                                                 NRRL B-18888                                                                              Sept. 19, 1991                                    Bacillus thuringiensis PS45B1                                                                 NRRL B-18396                                                                              August 16, 1988                                   Bacillus thuringiensis PS75J1                                                                 NRRL B-18781                                                                              March 7, 1991                                     Bacillus thuringiensis PS86A1                                                                 NRRL B-18400                                                                              August 16, 1988                                   Bacillus thuringiensis PS86Q3                                                                 NRRL B-18765                                                                              February 6, 1991                                  Bacillus thuringiensis                                                                        NRRL B-18812                                                                              April 23, 1991                                    PS140E2                                                                       Bacillus thuringiensis PS143J                                                                 NRRL B-18829                                                                              May 31, 1991                                      Bacillus thuringiensis                                                                        NRRL B-18832                                                                              May 31, 1991                                      PS202U2                                                                       Bacillus thuringiensis PS74G1                                                                 NRRL B-18397                                                                              August 16, 1988                                   Bacillus thuringiensis PS98A3                                                                 NRRL B-18401                                                                              August 16, 1988                                   Bacillus thuringiensis                                                                        NRRL B-18890                                                                              October 1, 1991                                   PS101Z2                                                                       ______________________________________                                    

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C.122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

The B.t. isolates of the invention can be cultured using standard artmedia and fermentation techniques. Upon completion of the fermentationcycle, the bacteria can be harvested by first separating the B.t. sporesand crystals from the fermentation broth by means well known in the art.The recovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules, or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests. These formulation and application procedures are all well knownin the art.

Formulated products can be sprayed or applied onto foliage to controlphytophagous beetles or larvae.

Another approach that can be taken is to incorporate the spores andcrystals of the B.t. isolates into bait granules containing anattractant and applying these granules to the soil for control ofsoil-inhabiting and foliage feeding Coleoptera. Formulated B.t. isolatescan also be applied as a seed-coating or root treatment or total planttreatment.

The B.t. isolates cells can be treated prior to formulation to prolongthe pesticidal activity when the cells are applied to the environment ofa target pest. Such treatment can be by chemical or physical means, orby a combination of chemical and/or physical means, so long as thetechnique does not deleteriously affect the properties of the pesticide,nor diminish the cellular capability in protecting the pesticide.Examples of chemical reagents are halogenating agents, particularlyhalogens of atomic no. 17-80. More particularly, iodine can be usedunder mild conditions and for sufficient time to achieve the desiredresults. Other suitable techniques include treatment with aldehydes,such as formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropyl and ethanol; varioushistologic fixatives, such as Bouin's fixative and Helly's fixative(See: Humason, Gretchen. L., Animal Tissue Techniques, W. H. Freeman andCompany, 1967); or a combination of physical (heat) and chemical agentsthat prolong the activity of the toxin produced in the cell when thecell is applied to the environment of the target pest(s). Examples ofphysical means are short wavelength radiation such as gamma-radiationand X-radiation, freezing, UV irradiation, lyophilization, and the like.

Genes encoding toxins having activity against the target susceptiblepests can be isolated from the B.t. isolates of the invention by use ofwell known procedures.

The toxin genes of the subject invention can be introduced into a widevariety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the situs of coleopteran insects where they willproliferate and be ingested by the insects. The result is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere"(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. geneexpressing the toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA.The initiation and translational termination region will involve stopcodon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the toxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a toxin gene islost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898,4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pRO1614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, Streptomyces lividans, and thelike.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the recombinant microbial cell can be done as disclosedinfra. The treated cells generally will have enhanced structuralstability which will enhance resistance to environmental conditions.Where the pesticide is in a proform, the method of inactivation shouldbe selected so as not to inhibit processing of the proform to the matureform of the pesticide by the target pest pathogen. For example,formaldehyde will crosslink proteins and could inhibit processing of theproform of a polypeptide pesticide. The method of inactivation orkilling retains at least a substantial portion of the bio-availabilityor bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown inany convenient nutrient medium, where the DNA construct provides aselective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the coleopteranpest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling,baiting, or the like.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

Example 1--Culturing the B.t. isolates of the invention

    ______________________________________                                        A subculture of a B.t. isolate can be used to inoculate the following         medium, a peptone, glucose, salts medium.                                     ______________________________________                                        Bacto Peptone           7.5 g/l                                               Glucose                 1.0 g/l                                               KH.sub.2 PO.sub.4       3.4 g/l                                               K.sub.2 HPO.sub.4      4.35 g/l                                               Salt Solution           5.0 ml/l                                              CaCl.sub.2 Solution     5.0 ml/l                                              Salts Solution (100 ml)                                                       MgSO.sub.4.7H.sub.2 O  2.46 g                                                 MnSO.sub.4.H.sub.2 O   0.04 g                                                 ZnSO.sub.4.7H.sub.2 O  0.28 g                                                 FeSO.sub.4.7H.sub.2 O  0.40 g                                                 CaCl.sub.2 Solution (100 ml)                                                  CaCl.sub.2.2H.sub.2 O  3.66 g                                                 pH 7.2                                                                        ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

Example 2--Testing of B.t. isolates Spores and Toxin Crystals Againstthe Rape Flea beetle, Egyptian Alfalfa Weevil, and Western SpottedCucumber Beetle

B.t. isolates of the subject invention were tested against the Rape Fleabeetle (Phyllotreta cruciferae) as follows:

Leaf sections were dipped into a suspension containing the B.t. isolateand allowed to dry. Adult beetles were placed in plasic cups, and theleaf section was placed between the cup rim and the cardboard lid. Cupswere inverted and held on moist blotter paper at room temperature. Fourdays after treatment the amount of leaf feeding damage was recorded.

B.t. isolates of the subject invention were also tested against theEgyptian alfalfa weevil (Hypera brunneipennis) as follows:

A suspension of the B.t. preparation was pipetted onto the surface of a1.5% agar diet in a 24 well assay tray. When it was completely dry, one,second instar, larvae was placed in each well. A sheet of Mylar was heatsealed over the tray and pierced with pins, and the tray was held at 25°C. for six days before mortality was determined.

B.t. isolates of the subject invention were further tested against theWestern Spotted Cucumber Beetle (Diabrotica undecimpunctataundecimpunctata) as follows:

Approximately 1 ml of 1.5% agar diet was added to each well of an assayplate. The surface of the diet was treated with a B.t. suspension,allowed to dry then infested with up to 5 larvae per well before thetray was sealed with ventilated Mylar. Mortality was determined atintervals, and a larval weight or instar determination was finally madeon day 10.

Example 3--Insertion of Toxin Gene Into Plants

The novel genes coding for the novel insecticidal toxins, as disclosedherein, can be inserted into plant cells using the Ti plasmid fromAgrobacter tumefaciens. Plant cells can then be caused to regenerateinto plants (Zambryski, P., Joos, H., Gentello, C., Leemans, J., VanMontague, M. and Schell, J [1983] Cell 32:1033-1043). A particularlyuseful vector in this regard is pEND4K (Klee, H. J., Yanofsky, M. F. andNester, E. W. [1985] BioTechnology 3:637-642). This plasmid canreplicate both in plant cells and in bacteria and has multiple cloningsites for passenger genes. A toxin gene, for example, can be insertedinto the BamHI site of pEND4K, propagated in E. coli, and transformedinto appropriate plant cells.

Example 4--Cloning of Novel B. thuringiensis Gene Into Baculoviruses

The novel genes of the invention can be cloned into baculoviruses suchas Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmidscan be constructed that contain the AcNPV genome cloned into acommercial cloning vector such as pUC8. The AcNPV genome is modified sothat the coding region of the polyhedrin gene is removed and a uniquecloning site for a passenger gene is placed directly behind thepolyhedrin promoter. Examples of such vectors are pGP-B6874, describedby Pennock et al. (Pennock, G. D., Shoemaker, C. and Miller, L. K.[1984] Mol. Cell. Biol. 4:399-406), and pAC380, described by Smith etal. (Smith, G. E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell.Biol. 3:2156-2165). The gene coding for the novel protein toxin of theinvention can be modified with BamHI linkers at appropriate regions bothupstream and downstream from the coding region and inserted into thepassenger site of one of the AcNPV vectors.

We claim:
 1. A process for controlling coleopteran pests selected fromthe group consisting of the genera Hypera, Diabrotica, and Phyllotretawhich comprises contacting said pests with an insect-controllingeffective amount of a Bacillus thuringiensis isolate selected from thegroup consisting of:PS92B having the identifying characteristics of NRRLB-18889, PS28Q2 having the identifying characteristics of NRRL B-18888,PS75J1 having the identifying characteristics of NRRL B-18781, PS140E2having the identifying characteristics of NRRL B-18812, PS143J havingthe identifying characteristics of NRRL B-18829, PS101Z2 having theidentifying characteristics of NRRL B-18890, or, a δ-endotoxin from oneof said isolates which kills a coleopteran insect of the genera Hypera,Diabrotica, or Phyllotreta.
 2. The process, according to claim 1,wherein said Hypera sp. is Hypera brunneipennis (Egyptian alfalfaweevil).
 3. The process, according to claim 1, wherein said Phyllotretasp. is Phyllotreta cruciferae (rape flea beetle).
 4. The process,according to claim 1, wherein said Diabrotica sp. is Diabroticaundecimpunctata undecimpunctata (corn rootworm).
 5. The process,according to claim 1, wherein said Bacillus thuringiensis isolate isBacillus thuringiensis PS92B.
 6. The process, according to claim 1,wherein said Bacillus thuringiensis isolate is Bacillus thuringiensisPS28Q2.
 7. The process, according to claim 1, wherein said Bacillusthuringiensis is Bacillus thuringiensis PS101Z2.
 8. A composition ofmatter comprising Bacillus thuringiensis isolate PS28Q2 having theidentifying characteristics of NRRL B-18888, or spores or crystals fromsaid isolate in association with an insecticide carrier or withformulation ingredients applied as a seed coating.
 9. A composition ofmatter comprising Bacillus thuringiensis isolate PS101Z2 having theidentifying characteristics of NRRL B-18890 or spores or crystals fromsaid isolate in association with an insecticide carrier or withformulation ingredients applied as a seed coating.
 10. A biologicallypure culture selected from the group consisting of Bacillusthuringiensis PS28Q2, having the identifying characteristics of NRRLB-18888, Bacillus thuringiensis PS92B, having the identifyingcharacteristics of NRRL B-18889 and Bacillus thuringiensis PS101Z2,having the identifying characteristics of NRRL B-18890.